CONFIDENTIAL 2012 Ingenza NewProt Kick-off meeting 25 Jan 2012 Nijmegen Ian Archer Ingenza Ltd Roslin, Edinburgh UK

CONFIDENTIAL 2012 Ingenza – what we do  Synthetic Biology Technologies  Biocatalysis/Bioprocess development  Protein expression/enzyme evolution  Screening for improved biocatalysts/processes  Novel enabling technologies e.g. gENABLE  Business areas  Fine chemicals  Biologics  Biofuels  Synthetic biology / petrochemical replacement  Biopolymers  Diagnostics

CONFIDENTIAL 2012 Platform technologies –e.g. oxidase and aminotransferase biocatalysts –Unnatural amino acids and chiral amines Large scale processes, compounds >99% e.e. –Highly expressed enzymes –High cell density fermentation –Efficient process chemistry Enzymes adapted by mutation/screening –New specificities –Improved reaction properties Process optimisation –Stabilised biocatalysts –Diverse enzyme production systems –Biocatalyst formulation –Statistical design of experiments Initial company focus: biosynthesis of chiral compounds Engineered microbial biocatalysts

CONFIDENTIAL 2012 Bacteria: E.coli –Large range of host/vector systems –Inducible, constitutive, synthetic C.glutamicum / C.acetobutylicum –Customising host/vector systems Yeast: Saccharomyces, Pichia –Range of host/vector systems –Cytosolic/Partitioned/Secretion –Integration vectors/copy number control –Fusions to identify novel regulatory regions Fungi: A.niger, A. terreus –Customising host/vector systems –Inducible/Constitutive Insect cell/mammalian –Production of biologics Expression systems Continuing to expand and diversify

CONFIDENTIAL 2012 Adapted to solid phase Proprietary Very high throughput –Millions of variants HRP Substrate oxidase Screening to adapt enzyme specificity or improve performance Colorimetric, qualitative solid phase/quantitative liquid phase Micro-titre plate assay Straight forward visual read-out Kinetic characterisation of variants

CONFIDENTIAL 2012 The screen detects – improved activity AND/OR –improved expression Can be used in conjunction with: Gene synthesis, now standard in molecular biology Gene assembly methods to rapidly generate expression libraries Assists selection of the optimal expression system Assists selection of the optimal gene sequence Delivers the highest quantity and quality of expressed protein –Screening libraries is a powerful tool Colorimetric screening Improved enzyme activity or protein expression

CONFIDENTIAL 2012 Stable in processStable in process –Resistant to chemical denaturation –Resistant to physical denaturation Not stable in processNot stable in process –Susceptible to chemical denaturation –Susceptible to physical denaturation Enzyme adaptation: Enzyme adaptation: Increased enzyme thermostability/robustness Three rounds of laboratory evolutionThree rounds of laboratory evolution

CONFIDENTIAL 2012 Library built (50,000 variants) Oversampled (500,000 clones) Hits identified visually 500 initial positive hits - re-assayed - PCR screen to confirm - liquid phase assay to quantify 50 Best hits identified - sub-cloned - SDS-PAGE/Western assay - Provided to customer Screening for efficient protein production Applied to biologic target to identify top 50 from 50,000 Over-expressing clone

CONFIDENTIAL 2012 Synthetic biology Replacement of petrochemical and other starting materials Partnership with Lucite International –Global producer of industrial polymers –Engaged in multi year contract –Microbial strain construction –Synthetic Biology – pathway engineering –Screening: Crossfeeding, Zone clearing, pH based –Fermentation development –Management of strategic academic collaboration Additional contracts now initiated –Biomass as replacement for petrochemical feedstock –Fermentation route to natural food additive –Multi-target –Synthetic Biology to develop efficient production microbes –Bacteria/Yeast/Fungi Proprietary genetic platforms accelerate strain improvement

CONFIDENTIAL 2012 gENABLE – Genome segment assembly Co-developed by Ingenza and Scottish Government Ingenza applying broadly in Industrial Biotechnology Assembly of genes, variants, reporters, markers, regulatory elements High-throughput, one-pot combinatorial assemblies Combines Bioinformatics, Microfluidics, Novel bio-reactions Accelerates: –Optimisation of gene expression –Pathway construction/engineering –Efficient synthesis of target products Applicable in all areas of protein expression Now central to all Ingenza enabling technologies and business areas Synergistic with screening Colorimetric, pH, crossfeeding, zone clearing, protein fusions

CONFIDENTIAL 2012 gENABLE Why? Expressing proteins is easy - isn’t it? Welch. M. Journal of the Royal Society. Interface 11 th March (2009)

CONFIDENTIAL 2012 Assemblies of up to 10 parts have been demonstrated gENABLE Specific linker based genetic pathway construction

CONFIDENTIAL 2012 Pr. 1 2 µ origin CEN4 origin Vector backbones Position APosition BPosition CPosition DPosition E Gene 1 Ter. Pr. 2 Gene 1 Ter. Pr. 3 Gene 1 Ter. Pr. 1 Gene 2 Ter. Pr. 2 Gene 2 Ter. Pr. 3 Gene 2 Ter. Pr. 1 Gene 3 Ter. Pr. 2 Gene 3 Ter. Pr. 3 Gene 3 Ter. Marker 3 variants 2 origins of replication 1 marker Assembly of 54 different genetic constructs in a single reaction Overcomes limits of empirical bioprocess optimisation Faster route to optimal bioprocess gENABLE 5 part combinatorial assembly for co-ordinated enzyme expression

CONFIDENTIAL 2012 Combination of 5 independent DNA fragments Assembly of synthetic enzyme pathway Typically a problematic empirical process Results in 95% success in correct pathway assembly (dark clones) > 95 % positive clones gENABLE Example of results

CONFIDENTIAL 2012 NewProt Workpackage Ingenza Experimental validation Tasks 1.Bioinformatics to identify aminotransferases (plus as many as possible of at least another 11 enzyme superfamilies) with diverse, novel activities towards commercially specific targets 2.Pathway engineering to incorporate activities into hosts 3.Develop screens to identify best production constructs 4.Screening of libraries to identify desired activities Focus areas? 1.Enzyme promiscuity to identify novel activities 2.Use of bioinformatics to demystify expression / activity etc